The present invention relates to biodetectors for detecting and quantifying molecules in liquid, gas, or on solid matrices. More specifically, the present invention relates to biodetectors comprising a molecular switching mechanism to express a reporter gene upon interaction with target substances. The invention further relates to methods using such biodetectors for detecting and quantifying selected substances with high specificity and sensitivity.
The detection of low-levels of biological and inorganic materials in biological samples, in the body or in the environment is frequently difficult. Assays for this type of detection involve multiple steps which can include binding of a primary antibody, several wash steps, binding of a second antibody, additional wash steps, and depending on the detection system, additional enzymatic and washing steps. Such assays further suffer from lack of sensitivity and are subject to inaccuracies. For instance, traditional immunoassays have false negative results of up to 30% when detecting infections.
Molecular probe assays, although sensitive, require highly skilled personnel and knowledge of the nucleic acid sequence of the organism. Both the use of nucleic acid probes and assays based on the polymerase chain reaction (PCR) can only detect nucleic acid which require complicated extraction procedures and may or may not be the primary indicator of a disease state or contaminant. Both types of assay formats are limited in their repertoire in cases where little information is available for the entity to be detected.
Current noninvasive methods to measure a patient""s physical parameters, such as CAT or MRI, are expensive and are often inaccessible. Thus, the monitoring of many medical problems still requires tests, which can be slow and expensive. The time between the actual test and the confirmation of the condition may be very important. For example, in the case of sepsis, many patients succumb before infection is confirmed and the infecting organism identified, thus treatment tends to be empirical and less effective. Another example is in screening the blood supply for pathogens.
Verification of a pathogen free blood supply requires a number of labor intensive assays. In the case of HIV-1, the virus that causes AIDS, the current assays screen for anti-HIV antibodies and not the virus itself. There is a window lasting up to many weeks after exposure to the virus in which antibodies are not detectable, and yet the blood contains large amounts of infectious virus particles. Clark et al., 1994, J. Infect. Dis. 170:194-197; Piatak et al., 1993, Aids Suppl. 2:S65-71. Thus, screening of the blood supply is not only time-consuming and slow, it may also be inaccurate.
Similarly, the ability to detect substances in the environment, such as airborne and waterbome contaminants is of great importance. For example, it would be desirable to monitor groundwater, to control industrial processes, food processing and handling in real-time using an inexpensive versatile assay. However, current methods are not suited for such xe2x80x9con-linexe2x80x9d monitoring.
There are several reasons why current methods are limited. First, access to sufficient amounts of the material to be detected may be difficult. For example, the detection of biological materials can be difficult as the biological materials of interest are often sequestered inside a body, and large quantities can be difficult to obtain for ex vivo monitoring. Therefore, sensitive assays for use on small amounts of material are necessary. This indicates that a method of amplifying the signal is required. Amplification methods have been established for detection of nucleic acids but this is not the case for antigen detection methods.
A second problem is that sensing may be difficult in real-time because the target materials may be present in such small quantities that detection of their presence requires time-consuming, expensive and technically-involved processes. For example, in the case of bacterial infections in the blood, sepsis, there may be only 1-2 bacteria in a 1-10 ml blood sample. Current methods require that the bacteria are grown first in order to be detected. Askin, 1995, J. Obstet. Gynecol. Neonatal. Nurs. 24:635-643. This time-lag may be detrimental as delaying treatment or mistreating diseases may mean the difference between life and death.
Others have attempted to avoid these limitations by using radioactive or fluorescent tags in combination with antibodies (Harlow et al., (1988), Antibodies. A Laboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Antibody-based assays typically involve binding of a primary antibody to the target molecule, followed by a series of washing steps to remove all unbound antibodies. Specific binding is typically detected using an identifier molecule, such a labeled secondary antibody directed against the primary antibody. This step is also followed by multiple wash steps. Alternatively, the primary antibody may be directly attached to a detectable label. Suitable labels have included radioactive tracers, fluorescent tags, and chemiluminescent detection systems. Harlow, et al., 1988, Antibodies. A Laboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).
The series of steps required using such antibody-based assays to generate a specific signal are time consuming and labor intensive. Furthermore, these type of assays are limited to the detection of antigens fixed to some type of matrix. Examples of this type of detection system include Western blots, immunohistochemistry, and ELISA. The highest sensitivity is currently achieved using radioisotopic and chemiluminescent tags. However, sensitivity, i.e., specific signal over background, of these detection systems frequently remains a limiting factor.
Similarly, background radiation places limits on the sensitivity of radioactive immunoassay techniques. In addition, these techniques are time-consuming and expensive. Finally, radioactive approaches are hostile to the environment, as they present significant waste disposal problems.
Another approach to monitoring substances involves the use of light. Light has the advantage that it is easily measurable, noninvasive and quantitative. Von Bally et al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag).
Traditional spectroscopy involves shining light into substances and calculating concentration based upon the absorbance or scattering of light. Von Bally et al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag). Optical techniques detect variations in the concentration of light-absorbing or light scattering materials. Von Bally et al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag). Near-infrared spectroscopy has proved to be a relatively safe form of radiation that functions well as a medical probe, since it can penetrate into tissues. Further, it is well-tolerated in large dosages. For example, light is now used to calculate the concentration of oxygen in the blood (Nellcor) or in the body (Benaron image), or even to monitor glucose in the body (Sandia). Benaron and Stevenson, 1993, Science 259:1463-1466; Benaron et al., 1993, in: Medical Optical Tomography: Functional Imaging and Monitoring, G. Muller, B. Chance, R. Alfano and e. al., eds. (Bellingham, Wash. USA: SPIE Press), pp. 3-9; Benaron and Stevenson, 1994, Adv. Exp. Med. Biol. 361:609-617. However, current techniques are limited in that many substances do not have unique spectroscopic signals which can be optically assessed easily and quantitatively. Von Bally et al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag). Furthermore, the detection of substances at low concentration is frequently hampered by high background signals, especially in biological media such as tissues. Von Bally et al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag).
Over the past years, assays based on light emission, for example chemiluminescence (Tatsu and Yoshikawa, 1990, Anal. Chem. 62:2103-2106), have attracted increasing attention due to the development of extremely sensitive methods for detecting and quantifying light. Hooper et al., 1994 J. Biolumin. Chemilumin. 9:113-122. One example of a biomedical research product using chemiluminescence is the ECL detection system (Amersham) for immunoassays and nucleic acid detection.
The use of biological sources of light, bioluminescence, for biological assays has paralleled development of chemiluminescent detection, as similar devices for light detection are required. Kricka, 1991, Clin. Chem. 37:1472-1481. One of the most commonly employed biological sources of light is luciferase, a light-generating enzyme synthesized by a range of organisms, including Photinus pyralis (American firefly), Renilla reniformis (phosphorescent coral), and Photobacterium (luminescent bacterial species). Generally, luciferase is a low molecular weight oxidoreductase, which catalyzes the dehydrogenation of luciferin in the presence of oxygen, ATP and magnesium ions. During this process, about 96% of the energy released appears as visible light. For review, see, Jassim et al., 1990, J. Biolumin. Chemilumin. 5:115-122.
The sensitivity of photon detection and the ability to engineer bacteria and other cells to express bioluminescent proteins permit the use of such cells as sensitive biosensors in environmental studies. Guzzo et al., 1992, Toxicol. Lett. 64:687-693; Heitzer et al., 1994, Appl. Environ. Microbiol. 60:1487-1494; Karube and Nakanishi, 1994, Curr. Opin. Biotechnol. 5:54-59; Phadke, 1992, Biosystems 27:203-206; Selifonova et al., 1993, Appl. Environ. Microbiol. 59:3083-3090. For example, Selifonova et al. describe biosensors for the detection of pollutants in the environment. More specifically, using fusions of the Hg(II) inducible Tn21 operon with the promoterless luxCDABE from Vibrio fischeri, highly sensitive biosensors for the detection of Hg(II) have been constructed.
In addition to systems where bioluminescence is used as a detection method of a specific condition, e.g., the presence of Hg(II), supra, constitutive expression of luciferase has been employed as a marker to track viability of bacterial cells, as the luciferase assay is dependent on cell viability. For example, constitutive expression of luciferase has recently been employed in a bacterial disease model for testing of drugs and vaccines. Specifically, using an enhanced luciferase-expressing Mycobacterium tuberculosis strain has been employed to evaluate antibacterial activity in mice. Hickey et al., 1996, Antibacterial Agents and Chemotherapy 40:400-407.
However, currently-available biosensors are limited to the detection of those molecules for which an endogenous bacterial receptor exists. In contrast, the present invention enables the generation of biosensors selective for any antigen or substance which can be selectively recognized by an antibody or receptor. Specifically, the present invention combines the selectivity of ligand-specific binding and the versatility of the antibody repertoire with the sensitivity of bioluminescent detection, employing entities that specifically respond with photon emission to predetermined ligands. The approach of the present invention thus permits the generation of extremely sensitive biodetectors for the development of a wide variety of assays detecting any number of commercially important molecules.
The present invention is directed to targeted ligand-specific biodetectors for detecting and monitoring selected substances. More specifically, the biodetectors of the present invention comprise (1) a signal converting element, (2) a transducer, (3) a responsive element, and (4) a reporter gene. The signal converting element comprises an extracellular ligand-specific moiety and an intracellular signal transforming domain. The extracellular ligand-specific moiety specifically recognizes a selected substance. Recognition of the substance by the extracellular ligand-specific moiety activates the intracellular signal transforming domain. The activated signal-transforming domain in turn activates the transducer, which in its activated form is capable of binding to and activating the responsive element. The responsive element, typically a promoter, is operatively linked to the reporter gene, which encodes a polypeptide with unique properties that are easily detected, e.g., optically. Thus, the biodetectors of the invention convert the action of binding to a target substance, i.e., a ligand, into a detectable signal.
In a general embodiment, the signal converting element is a fusion protein where the extracellular ligand-specific moiety and the intracellular signal transforming domain are heterologous to one another (i.e., are derived from different proteins). In a preferred embodiment, the extracellular ligand-specific moiety is an antibody fragment, such as a single chain variable fragment (ScFv).
In yet another embodiment, of the invention, the intracellular signal transforming element is derived from a membrane signal sensor molecule. The membrane signal sensor may be selected from the group consisting of the xe2x80x9csensorxe2x80x9d of a bacterial two component regulatory system, a eukaryotic receptor, and a prokaryotic receptor. In a specific embodiment, the intracellular signal transforming domain comprises the 3xe2x80x2 end of the phoQ gene, which encodes the active or signal transforming portion of PhoQ, the xe2x80x9csensorxe2x80x9d of the PhoQ/PhoP bacterial two component regulatory system. In a more specific embodiment, the intracellular signal transforming domain comprises the cytoplasmic tail of PhoQ, defined as amino acids 219-487 of the PhoQ polypeptide sequence. In another specific embodiment, the intracellular signal transforming domain comprises the cytoplasmic tail of PhoQ along with the immediately adjacent transmembrane segment, together defined as amino acids 190-487 of the PhoQ polypeptide sequence (Miller, et al., 1989, Proc Natl Acad Sci 86:5054-5058).
In another embodiment, the signal converting element further comprises a membrane anchor positioned between the between the extracellular ligand-specific moiety and the intracellular signal transforming domain. In a specific embodiment, the membrane anchor is derived from E. coli cell envelope component PAL. In yet another embodiment, the signal converting element further comprises an N-terminal leader sequence positioned upstream of the extracellular ligand-specific moiety.
In another general embodiment, the responsive element comprises a transcription control element which is activated by the active form of the transducer. One embodiment of the invention thus includes promoters and/or transcription activators which control transcription in two-component systems. In a particular embodiment, the responsive element comprises the phoN promoter.
In still another general embodiment, the biodetector comprises an intact bacterial cell transfected as detailed herein. In one embodiment, the biodetector is a Gram-positive bacterial cell. Such a biodetector may be selected from the group consisting of Streptococcus, Staphylococcus, Listeria, Clostridium, Bacillus, Tuberculosis, and Corynebacteria. In another embodiment, the biodetector is a Gram-negative bacterial cell. Such a biodetector may be selected from the group consisting of Escherichia, Salmonella, Pseudomonas, Helicobacter, Shigella, Proteus, Bordetella, Neisseria, Haemophilus, Bacteriodes, Vibrio, Brucella, Campylobacter, Rickettsia, Enterococci, Klebsiella, Spirochetes, and Yersinia. Preferred embodiments of Gram negative biodetectors comprise Escherichia and Salmonella.
In still another embodiment of the invention, the substance that the biodetector is designed to detect is selected from the group consisting of bacteria, bacterial products, viruses, protein, sugar, lipid, liposaccharide and polysaccharide.
In another aspect, the invention includes a biodetector for the detection of a selected substance. The biodetector comprises (a) a signal converting element, comprising an extracellular ligand-specific moiety and an intracellular signal transforming domain, wherein the extracellular ligand-specific moiety specifically recognizes the selected substance, which recognition activates the intracellular signal transforming domain; (b) a transducer, wherein the transducer has an inactive and an active form which are distinct from each other, and wherein the activated intracellular signal transforming domain converts the inactive form of the transducer into the active form of the transducer; and (c) a responsive element, wherein the responsive element is bound and activated by the active form of the transducer, resulting in a detectable signal.
In one embodiment, the responsive element further comprises a nucleic acid encoding one or a plurality of gene product(s), which gene product or gene products produce the detectable signal, and wherein the nucleic acid is operatively linked to the transcription control element. In a more specific embodiment, the detectable signal is visible light. In another specific embodiment, the gene product is detectable by means selected from the group consisting of bioluminescence, colorimetric reactions and fluorescence. In a more specific embodiment, the gene product is detectable by means of bioluminescence. In yet a more specific embodiment, the nucleic acid comprises a luciferase operon.
Other specific embodiments of this aspect of the invention include those summarized above for other biodetectors made in accordance with the invention.
Also included in the invention is a library of biodetectors, where the biodetectors have characteristics, in different specific embodiments of such a library, as described above. In a general embodiment, the biodetectors in such a library comprise a plurality of bacterial cells transfected with a mixture of cDNA molecules encoding antibody variable region genes, Fab fragments, F(abxe2x80x2)2 fragments, or single chain variable fragments (ScFvs). The library preferably includes at least about 1000 different biodetectors, more preferably at least about 10,000 different biodetectors, and even more preferably at least about 100,000 different biodetectors.
The present invention is further directed to methods of using such biodetectors for detecting and monitoring selected substances with a high sensitivity and specificity (selectivity). The methods using the biodetectors of the invention include the detection of contaminants in the food and agriculture industries, diagnosis and monitoring in medicine and research, detection of poisons or pathogenic contaminants in the environmental or defense setting, and drug testing. Accordingly, the invention includes a method for detection of a selected substance. The method comprises the steps of (a) generating a biodetector; (b) adding the biodetector to a sample; (c) measuring and quantifying the detectable signal; and correlating the levels of the detectable signal with the presence and quantity of the substance. The biodetector generated in step (a) comprises (i) a signal converting element, comprising an extracellular ligand-specific moiety and an intracellular signal transforming domain, wherein the extracellular ligand-specific moiety specifically recognizes the selected substance, which recognition activates the intracellular signal transforming domain; (ii) a transducer, wherein the transducer has an inactive and an active form which are distinct, and wherein the inactive form is converted into the active form by the activated intracellular signal transforming domain; and (iii) a responsive element, wherein the responsive element is bound and activated by the active form of the transducer, resulting in a detectable signal.
In one embodiment, the responsive element of the biodetector in the method comprises a transcription control element which is activated by the active form of the transducer. In another embodiment, the responsive element further comprises a nucleic acid encoding one or a plurality of gene products which gene product or gene products produce the detectable signal, and wherein the nucleic acid is operatively linked to the transcription control element. In a preferred embodiment, the detectable signal is light. The gene product in such an embodiment may be detected by a means selected from the group consisting of bioluminescence, calorimetric reactions and fluorescence. In a specific embodiment, the nucleic acid comprises a luciferase operon. In a more specific embodiment, the light detection system is selected from the group consisting of luminometer, spectrophotometer, fluorimeter, and a CCD detector.
In another embodiment, the biodetector or the sample in the method is fixed on a solid support. In yet another embodiment, the method further includes fixing a series of biodetectors in an ordered array on a solid support such that a variety of substances comprised in a sample can be detected.
In another aspect, the invention includes an expression vector useful for making a biodetector. The vector comprises (i) a cloning site for insertion of a DNA fragment encoding an extracellular ligand-specific moiety, and (ii) a first DNA fragment encoding an intracellular signal transforming domain. Preferably, the vector is capable of expressing a fusion protein comprising (a) a polypeptide encoded by a DNA sequence inserted at said cloning site, and (b) the intracellular signal transforming domain. In one embodiment, the vector further comprises, between the cloning site and the first DNA fragment, a second DNA fragment encoding a membrane anchor. In another embodiment, the vector further comprises, upstream of the cloning site, a third DNA fragment encoding an N-terminal leader sequence. In another embodiment, the vector further comprises, inserted at the cloning site, a fourth DNA fragment encoding an extracellular ligand-specific moiety. In another embodiment, the extracellular ligand-specific moiety comprises an antibody fragment. In another embodiment, the first DNA fragment encodes a polypeptide comprising the cytoplasmic tail of PhoQ.